1. Volume 17, Issue 2, Pages 221-232 (August 2015)
CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library 
Cuiqing Zhong, Qi Yin, Zhenfei Xie, Meizhu Bai, Rui Dong, Wei Tang, Yu-Hang Xing, Hongling Zhang, Suming Yang, Ling-Ling Chen, Marisa S. Bartolomei, Anne Ferguson-Smith, Dangsheng Li, Li Yang, Yuxuan Wu, Jinsong Li 
Cell Stem Cell 
Volume 17, Issue 2, Pages (August 2015)
DOI: /j.stem
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2. Cell Stem Cell 2015 17, 221-232DOI: (10.1016/j.stem.2015.06.005)
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3. Figure 1
AG-haESCs Harboring Both H19ΔDMR and IGΔDMR Efficiently Support the Generation of Live SC Pups
(A) Diagram of SC mice generated by ICAHCI using AG-haESCs carrying H19- and IG-DMR deletions. The sperm injected into enucleated oocytes carried a deletion of H19-DMR. PPN, pseudopronucleus derived from injected haESCs.
(B) Image of androgenetic embryos developed from an injection of H19ΔDMR sperm into enucleated oocytes. A black arrow indicates a blastocyst, which had been used for ESCs derivation. Scale bar, 50 μm.
(C) Phase-contrast image of ESCs derived from one androgenetic blastocyst. Scale bar, 100 μm.
(D) Establishment of H19ΔDMR -AGH cell lines (represented by H19ΔDMR -AGH-1) after multiple rounds of FACS enrichment for haploid cells. A DAPI filter was used to detect the signal of Hoechst-stained DNA. The left panel shows FACS data of diploid control ESCs for comparison.
(E) SC pups from ICAHCI using H19ΔDMR -AGH-3 cells (passage 8). Pups and placentas obtained by C-section from a pseudopregnant mouse at E19.5 are shown.
(F) Schematic of sgRNAs targeting for removal of IG-DMR. Dark green bar represents the deleted region (4.15 kb). The sequences of IG-DMR-sgRNA1 and IG-DMR-sgRNA2 are indicated.
(G) Generation of H19ΔDMR- IGΔDMR-AGH cells. Left, mCherry-positive cells (3.52%), which were CRISPR-Cas9-transfected cells, were enriched and plated for derivation of AG-haESCs. Right: one established AG-haESC line (represented by H19ΔDMR- IGΔDMR-AGH-4).
(H) SC pups from ICAHCI using H19ΔDMR- IGΔDMR-AGH-4 cells (passage 29). Note that SC pups from H19ΔDMR- IGΔDMR-AGH-4 cells were naturally delivered by mothers.
See also Figures S1 and S2 and Table S1.
Cell Stem Cell  , DOI: ( /j.stem )
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4. Figure 2
H19 and IG DMRs Are Two Barriers to the High-Efficiency Generation of SC Pups in AG-haESCs
(A) SC pups from IGΔDMR-AGH-1 cells (passage 17). Pups and placentas obtained by C-section from a pseudopregnant mouse at E19.5 are shown. Asterisks indicate growth-retarded SC pups that died shortly after birth.
(B) Methylation state of the H19-DMR in a normal SC pup (left) and a retarded pup (right) derived from IGΔDMR-AGH-1 cells.
(C) Schematic of sgRNAs targeting for the removal of H19-DMR. A pink bar represents the deleted region (3.8 kb). The sequences of H19-DMR-sgRNA1 and H19-DMR-sgRNA2 are indicated.
(D) Genotyping analysis of H19ΔDMR-IGΔDMR-AGH-OG3 cells. Note that these cells were generated by deletion of both IG-DMR and H19-DMR in WT AGH-OG-3 (passage 21) that have lost the ability to produce SC pups after injection into oocytes (Yang et al., 2012).
(E) Genotyping analysis of the progeny of SC mice derived from DKO-AG-haESCs. Note that pups carrying mutant IG-DMR or mutations in both H19 and IG died shortly after birth.
(F) Gene expression profiles of DKO-AG-haESCs using RNA-seq analysis. Gene expression profiles were clustered using all expressed genes. Three DKO-AG-haESC lines generated by different strategies show highly similar expression profiles to the control AG-haESCs, although they are markedly different from mouse round spermatids (RSs). AGH-2 is a WT haploid cell line established in this study. AGH-OG-3 is a WT haploid cell line that was established in our previous study (Yang et al., 2012). To avoid the influence of diploidized cells on the expression profile, we collected samples after FACS of cells in the G1/G0 phase.
(G) Gene expression profiles of DKO-AG-haESCs based on imprinting genes. Three DKO-AG-haESC lines show highly similar expression profiles to the control AG-haESCs, although they are markedly different from mouse RSs.
(H) Methylation profiles of DKO-AG-haESCs based on RRBS analysis. The pies show different methylation levels of CpG sites in different colors.
See also Figures S2 and S3 and Table S1.
Cell Stem Cell  , DOI: ( /j.stem )
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5. Figure 3
DKO-AG-haESCs Carrying Multiple Gene Modifications Support Efficient Generation of SC Mice
(A) Schematic of Cas9-sgRNA-targeting sites in Tet1, 2, and 3. The sgRNA-targeting sequences are underlined, and the PAM sequences are labeled in red.
(B) MCherry-positive cells (3.96%), which were CRISPR-Cas9-transfected cells, were enriched and plated for derivation of DKO-AG-haESCs carrying mutant Tet genes.
(C) Generation of Tet-TKO-DAH cells. Left: phase-contrast image of ESCs derived by expansion of single cells. Right: flow analysis of an established haploid cell line. Scale bar, 100 μm.
(D) The sequences of Tet1, 2, and 3 in Tet-TKO-DAH-1 cells. Deletions are indicated with (−).
(E) DNA sequences of PCR products amplified from Tet1, 2, and 3 genes of two SC pups generated from Tet-TKO-DAH-1 and Tet-TKO-DAH-2, respectively. Two peaks can be observed in the sequences of the SC pups carrying heterogeneous mutations in Tet1, 2, and 3.
(F) Schematic of Cas9-sgRNA-targeting sites in p53, 63, and 73. The sgRNA-targeting sequences are underlined, and the PAM sequences are labeled in red.
(G) The sequence of p53, 63, and 73 in p53-TKO-DAH-2. Deletions are indicated with (−).
(H) Schematic of double-stranded vectors, including Tet1-EGFP, Tet2-mCherry, and Tet3-ECFP. EGFP, mCherry, and ECFP reporters fused with the last codon of the Tet1, Tet2, and Tet3 genes, respectively.
(I) Genotyping analysis of Tet-TKI-DAH-1 cells.
See also Figures S4 and S5 and Table S3.
Cell Stem Cell  , DOI: ( /j.stem )
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6. Figure 4
DKO-AG-haESCs Carrying an sgRNA Library Support the Efficient Generation of Heterozygous Mutant Mice in One Step
(A) Schematic of the efficient generation of heterozygous mutant SC mice via ICHACI using DKO-AG-haESCs carrying an sgRNA library in one step.
(B) Haploid cells expressing mCherry (15.8%), in which Cas9 were successfully transfected, enriched, and used for subsequent ICAHCI analysis.
(C) PCR analysis of sgRNA in haploid cell clones expanded from single cells. All tested clones carried sgRNA.
(D) The sequences of different targeted genes in cell clones. Gene modifications existed in the targeted genes in all tested clones.
(E) SC pups from DKO-AG-haESCs carrying an sgRNA library. Note that SC pups were naturally delivered by mothers.
(F) PCR analysis of existence of sgRNA in SC pups.
(G) The sequences of different targeted genes in SC pups. Gene modifications existed in the targeted genes in tested SC pups.
See also Data S1.
Cell Stem Cell  , DOI: ( /j.stem )
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7. Figure 5
DKO-AG-haESCs Carrying Constitutively Expressed Cas9 and an sgRNA Library Support the Efficient Generation of Biallelic Mutant SC Mice in One Step
(A) Schematic of efficient generation of biallelic mutant SC mice via ICHACI using DKO-AG-haESCs carrying constitutively expressed Cas9 and an sgRNA library.
(B) PCR analysis of Cas9 in haploid cell clones expanded from single cells. All tested clones carried Cas9 transgene.
(C) qPCR analysis of Cas9 expression in cell clones expanded from single cells. Cas9 were expressed in all tested cell clones.
(D) PCR analysis of sgRNA in haploid cell clones expanded from single cells. All tested clones carried sgRNA.
(E) SC pups from DKO-AG-haESCs carrying constitutively expressed Cas9 and an sgRNA library. Note that SC pups were naturally delivered by mothers. Left: newborn SC pups. Right: adult SC mice.
(F) Identification of sgRNA in SC mice by PCR analysis.
(G) Generation of biallelic mutant mice via ICAHCI using DKO-AG-haESCs carrying constitutively expressed Cas9 and an sgRNA library. One represented SC mouse carries biallelic mutant Polm gene, indicated by multiple peaks in the sequence of PCR products.
(H) Sequence of the targeted Polm gene in the mouse tail by TA cloning and sequencing analysis. 24 of 26 tested clones carried frameshift insertion/deletion (indel) mutations.
(I) Summary of TA cloning and sequencing analysis of seven biallelic mutant mice. Over 80% of tested clones carried frameshift indels.
(J) TA cloning and sequencing analysis of different organs in one mouse carrying biallelic mutant Scube1 gene. Over 80% of tested clones carried frameshift indel mutations. More than 30 clones were tested for each organ.
See also Figure S6, Table S4, and Data S1.
Cell Stem Cell  , DOI: ( /j.stem )
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